JP2833181B2 - FR901375 substance and production method thereof - Google Patents

Description

The present invention relates to novel biologically active compounds, hereinafter referred to as FR901375 substances. More specifically, the present invention relates to a novel and biologically active FR901375 substance having antibacterial and antitumor activities, a method for producing the same, and a pharmaceutical composition containing the same.

The FR901375 substance of the present invention is a substance belonging to the genus Pseudomonas, such as Pseudomonas chlororafis No. 2522.
It can be produced by culturing the R901375 substance producing strain in a nutrient medium.

 This fermentation process is described in detail below.

(1) Microorganisms Details of the microorganisms used for producing FR901375 substance are described below.

Taxonomic study of No. 2522 strain: No. 2522 strain was isolated from a soil sample collected in Chiba Prefecture.

For this taxonomic study, the methods described in the Barges Manual of Systematic Bacteriology (Vol. 1) were mainly employed.

(I) Morphological characteristics: Morphological observation of No. 2522 strain was performed on nutrient broth and agar
The cells cultured at 0 ° C. for 20 hours were examined with an optical microscope and an electron microscope.

The No. 2522 strain is a gram-negative bacterium, and exhibited motility due to polar trichome flagella. The cells are rod-shaped and have a size of about 0.
It was 7-0.8 × 2.0-2.5 μm.

This strain produced a diffusible fluorescent dye on Bacto pseudomonas agar F medium (manufactured by Difco). This strain produced a green soluble pigment (chlororafine), which crystallized in the culture medium in and around the colony.

 The results are shown in Table 1.

(Ii) Physiological characteristics: Table 2 summarizes the physiological characteristics of No. 2522 strain. The growth temperature range was 3 ° C to 35 ° C.

The No. 2522 strain was oxidase positive and catalase positive, and the OF test was oxidizing. This strain hydrolyzed gelatin and casein. Starch hydrolysis was negative. D-glucose, D-xylose, D
-Acid formation was observed from fructose, D-galactose, D-mannitol and sucrose. No acid was formed from lactose and maltose. This strain did not utilize L-arabinose and sorbitol.

(Iii) Identification Barges Manual of Systematic
According to bacteriology (Vol. 1), strain No. 2522 was considered to belong to the genus Pseudomonas from the above-mentioned various properties.

Barges Manual of Systematic
For a more detailed comparison, Pseudomonas chlororafis Guignard and Sauvageau 1984 were selected for a more detailed comparison only by comparing the properties of the Pseudomonas species described in Bacteriology (Vol. 1).

Therefore, the No.2522 strain was compared with Pseudomonas chlororafis. No significant difference was observed between the two cultures, and the properties of strain No. 2522 were in good agreement with Judemonas chlororafis.

Therefore, strain No. 2522 was identified as Pseudomonas chlororafis.

A culture of Pseudomonas chlororafis No. 2522 has been deposited under the deposit number FERM-BP2572 at the Research Institute of Microbial Industry, Institute of Industrial Science (1-3-1 Higashi, Tsukuba, Ibaraki Prefecture) (Deposit Date: 1989) August 29).

(2) Production of FR901375 substance The FR901375 substance of the present invention is useful for producing a FR901375 substance producing strain belonging to the genus Pseudomonas (for example, Pseudomonas chlororafis No. 2522) in a nutrient medium containing an assimilable carbon source and a nitrogen source. It is produced when grown under aerial conditions (eg, shaking culture, submerged culture, etc.).

Preferred carbon sources in the nutrient medium are carbohydrates such as glucose, starch, fructose, glycerin.

Preferred nitrogen sources are bouillon, yeast extract, peptone, gluten flour, cottonseed flour, soy flour, corn steep liquor, dried yeast, wheat malt and the like, as well as ammonium salts (such as ammonium nitrate, ammonium sulfate, ammonium phosphate etc.), urea, amino acids And inorganic and organic nitrogen compounds.

The carbon and nitrogen sources are advantageously used in combination, but need not be used in pure form, and low purity materials containing trace amounts of growth factors and substantial amounts of inorganic nutrients are also suitable for use.

If desired, inorganic salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, and cobalt salts may be added to the medium.

If necessary, especially when the medium foams violently, an antifoaming agent such as liquid paraffin, fatty oil, vegetable oil, mineral oil, silicone or the like may be added.

As with the method preferably used for mass production of other bioactive substances, submerged (deep) culture conditions are preferred for mass production of FR901375 substances.

 For small-scale production, shake culture in a flask is used.

In addition, when growing in a large tank, FR9013
It is preferred to use the vegetative cells of the bacterium in inoculating the production tank in order to avoid growth delays in the 75 substance production process. Therefore, first, it is desirable to inoculate a relatively small amount of medium with the cells of the bacterium, culture the inoculated medium to produce vegetative cells of the bacterium, and then transfer the cultured vegetative cells to a large tank. . The medium for producing the vegetative cells may be substantially the same as or different from the medium used for producing the FR901375 substance.

Agitation and aeration of the culture mixture can be performed by various methods. The stirring is performed by rotating or shaking the fermenter by a propeller or a similar mechanical stirring device.
This can be done by various pumping devices or by passing sterile air through the medium. Aeration can be performed by passing sterile air through the fermentation mixture.

Fermentation is usually carried out at a temperature between about 15 ° C and 35 ° C for about 15-50
This is done over time and these may vary depending on fermentation conditions and scale.

After completion of the fermentation, the culture solution is subjected to various steps conventionally used for the recovery and purification of the biologically active substance, for example, solvent extraction using a suitable solvent or a mixture of several solvents, chromatography or a suitable solvent or The FR901375 material is recovered by recrystallization from a mixture of several solvents.

In accordance with the present invention, the FR901375 material is generally found primarily in bacterial cells. However, the filtrate was also FR901375
Contains substance. Therefore, it is preferable to directly subject the entire culture to the FR901375 substance isolation step by extraction using a suitable solvent such as hot water, acetone, ethyl acetate or a mixture of these solvents.

The extract is processed in a conventional manner to obtain FR901375 substance.
For example, the extract is concentrated by evaporating or distilling it to a small volume, and the resulting residue containing the active substance (ie, FR901375 substance) is purified by a conventional purification method such as chromatography or a suitable solvent or several solvents. Purify by recrystallization from the mixture.

(3) Physicochemical properties of FR901375 substance: FR901375 substance obtained according to the above fermentation method has the following physicochemical properties.

Appearance: Colorless prismatic acid, neutral, basic: neutral substance Melting point: 222-226 ° C (decomposition) Specific rotation: ▲ [α] ²¹ _D ▼: -25.3 ゜ (c = 1.0, CHCl ₃ ) Molecular formula: C ₂₄ H ₃₈ N ₄ O ₇ S ₂ Elemental analysis value Calculated value for C ₂₄ H ₃₈ N ₄ O ₇ S ₂ : C, 51.59; H, 6.86; N, 10.03:
S, 11.48 (%) Found: C, 50.69; H, 6.78; H, 9.73; S, 11.47 (%) Molecular weight: 558 FAB-MS m / z 559 (M + H) ⁺ High-resolution FAB-MASS 559.2270 (C ₂₄ H ₃₈ N ₄ O ₇ S ₂ Calculated value: 559.2260) Solubility: Soluble: chloroform, ethyl acetate Slightly soluble: methanol, acetone Insoluble: water, hexane Color reaction: positive: cerium sulfate reaction, iodine vapor reaction Negative: Ninhydrin reaction, ferric chloride reaction, Ehrlich reaction, Maurish reaction Thin layer chromatography: UV absorption spectrum: Terminal absorption (in methanol) ¹ H nuclear magnetic resonance spectrum: [CD ₃ OD, 400 MHz] δ: 5.87 (1 H, m), 5.68 (1 H, broad d, J = 16 Hz), 5.60 (1
H, broad), 4.69 (1H, t, J = 5.5Hz), 4.43 (1H, d, J = 2.5H)
z), 4.33 (1H, dq, J = 2.5and6.5Hz), 4.02 (1H, d, J = 8.5
Hz), 3.56 (1H, d, J = 9.5Hz), 3.38 (2H, d, J = 5.5Hz),
2.95 (2H, m), 2.87 (1H, dd, J = 6.5and14Hz), 2.62 (1H,
dd, J = 7and14Hz), 2.61 (2H, m), 2.45 (1H, m), 2.09 (1
H, m), 1.17 (3H, d, J = 6.5Hz), 1.03 (3H, d, J = 6.5Hz),
1.01 (3H, d, J = 6.5Hz), 0.99 (3H, d, J = 6.5Hz), 0.98
(3H, d, J = 6.5 Hz) ppm ¹³ C nuclear magnetic resonance spectrum: (CD ₃ OD, 100 MHz) δ: 175.27 (s), 174.40 (s), 172.37 (s), 171.87
(S), 170.37 (s), 133.76 (d), 127.37 (d), 71.3
5 (d), 68.39 (d), 66.48 (d), 62.03 (d), 60.08
(D), 55.01 (d), 41.71 (t), 40.42 (t), 38.38
(T), 34.63 (t), 31.47 (d), 29.32 (d), 20.26
(Q), 20.19 (q), 19.93 (q), 19.86 (q), 19.08
(Q) ppm From the above physicochemical properties, FR901375 substance was proved to have the following chemical formula: (4) Biological properties of FR901375 substance: As an example showing the biological activity of FR901375 substance, some biological data will be described below.

Test Example 1 Antibacterial activity of FR901375 substance: The antibacterial activity of FR901375 substance was determined in Sabouraud medium by agar dilution method.

The minimum inhibitory concentration (MIC) is expressed in μg / ml after incubation at 25 ° C. for 48 hours. The results are shown in Table 3.

Test Example 2 Inhibition of Human Tumor Cell Proliferation In Vitro by FR901375 Substance In Vitro 4 × 10 ^{3 in} Dulbecco's minimum essential medium 100 μl supplemented with 10% fetal bovine serum, penicillin (50 units / ml) and streptomycin (50 μg / ml) Cytotoxicity tests were performed using microtiter plates containing individual tumor cells in each well. The cells were incubated at 37 ° C. for 4 days and the method described by Mosmann (JI
mmunol.Methods, ₆₅ , 55-63, 1983).
[3- (4,5-dimethylthiazol-2-yl) -2,5-
Diphenyltetrazolium bromide] assay was performed. MTT was dissolved in phosphate buffer (PBS) at a concentration of 5 mg / ml, and sterilized by filtration. At the same time, a small amount of insoluble material was removed. After culturing human tumor cells, the MTT solution (10 μM per 100 μ of medium) was added. ) Was added to all assay wells and the plates were further incubated at 37 ° C. for 4 hours. Acidic isopropanol (100 μl of 0.04 N HCl in isopropanol) was added to all wells and mixed well to dissolve the dark blue crystals. After all the crystals were dissolved, the plate was read at 550 nm with a reference wavelength of 660 nm using a two-wavelength microplate photometer (MTP-22; Corona Electric Co., Katsuta). The compound of interest of the present invention was dissolved in methanol, diluted with Dulbecco's minimum required medium, and added to the culture to a final concentration of 1 μg / ml or less. The results are shown in Table 4.

Test Example 3 Antitumor activity of FR901375 substance against P388 mouse leukemia The antitumor activity of FR901375 substance was determined in a mouse tumor system. P338 leukemia cells (1 × 10 ⁶ ) were implanted intraperitoneally into BDF ₁ mice (female, 7 weeks old). From tumor cell inoculation
Twenty-four hours later, FR901375 substance was administered intraperitoneally. FR901375
Intraperitoneal administration of the substance was continued for a further 3 days, once a day. FR901375 substance was suspended in saline containing 0.5% methylcellulose. Control mice received a saline solution containing 0.5% methylcellulose intraperitoneally.

The results are shown in Table 5. The antitumor activity was calculated as the average survival time of each group, and the T / C% of the average survival time (100 × treatment group /
(Control group).

Test Example 4 Acute toxicity of FR901375 substance The acute toxicity of FR901375 substance when injected intraperitoneally into BDF ₁ mouse was 5.6 mg / kg.

From the above test results, it is recognized that FR901375 substance has biological activity.

(5) Pharmaceutical composition containing FR901375 substance The pharmaceutical composition of the present invention comprises an active ingredient in the form of a mixture with an organic or inorganic carrier or excipient suitable for external use, oral administration or parenteral administration. For example, in the form of a pharmaceutical preparation in liquid, semi-solid or solid form, containing the substance FR901375. The active ingredient may be mixed with, for example, tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and other usual non-toxic pharmaceutically acceptable carriers for use in dosage forms suitable for use. Can be. If necessary, adjuvants, stabilizers, thickeners, coloring agents and fragrances may be used. The FR901375 substance may be contained in the pharmaceutical composition in an amount sufficient to exert a desired antitumor effect on a disease process or condition.

When the composition is applied to humans, it is preferably by intravenous, intramuscular or oral administration. FR90 effective for treatment
The dose of 1375 substance will vary with the age and condition of each individual patient to be treated and will be present, but for tumor treatment, it is generally 0.1% FR901375 substance / kg human body weight for intravenous administration. ~ 100 mg daily dose of FR901375 substance 0.1 kg / kg human body weight for intramuscular administration, 0.1-100 mg daily dose of FR901375 substance 0.1 kg / kg human body weight for oral administration
A daily dose of 100100 mg is administered each.

(6) Examples: The following examples are provided to explain the present invention in more detail.

Example 1 In each of ten 500 ml Erlenmeyer flasks, glucose (1
%) And broth (1%)
Was injected and sterilized at 120 ° C. for 30 minutes. One platinum loop of slope culture of Pseudomonas chlororaffis No. 2522 was inoculated into each medium and cultured at 30 ° C. for 24 hours on a rotary shaker. The obtained culture was subjected to glucose (1%), glycerin (2%).
%), Bouillon (0.5%), soybean flour (1%), (NH ₄ ) ₂ S
O ₄ (0.1%), corn steep liquor (1%), MgSO ₄
・ Five 30-liter jar fermenters containing a medium (17 liters) containing 7H ₂ O (0.1%), calcium carbonate (0.2%), and adecanol (antifoam, trademark, manufactured by Asahi Denka Kogyo) (0.05%) And inoculated in advance at 120 ° C. for 30 minutes, inoculated, aerated at 20 liters / min, and
The cells were cultured at 30 ° C. for 48 hours with stirring at pm.

Isolation and purification: After termination of the culture, the culture (75 l) was mixed with an equal volume of acetone. The mixture was filtered using diatomaceous earth (3 kg), the filtrate (150 l) was evaporated under reduced pressure to a volume of 90 l, adjusted to pH 4.0 with 6N HCl and extracted with 90 l of ethyl acetate . This extraction operation was performed twice, and the extracts were combined. The ethyl acetate extract was concentrated under reduced pressure to a volume of 2.0 liters. After dehydration with anhydrous sodium sulfate, the ethyl acetate solution was converted to silica gel (silica gel 60,
(70-230 mesh, E. Merck) and the mixture was evaporated under reduced pressure. After evaporation of the solvent, the resulting powder was subjected to column chromatography on the same silica gel (1.0 liter) packed with n-hexane-ethyl acetate (1: 1 v / v). After washing with n-hexane-ethyl acetate (1: 1 v / v, 45 l), ethyl acetate (4.5 l) and ethyl acetate-acetone (1: 1 v / v) were used.
v, 4.5 liters). The fractions containing the target compound were combined and concentrated under reduced pressure to obtain an oily residue. The oily residue was mixed with silica gel 60 (70-230 mesh) and the mixture was slurried in ethanol (20 ml). After evaporation of the solvent, the resulting dry powder was loaded on silica gel 60 (70-23
(0 mesh, 400 ml). The column was developed with chloroform (1 liter) and a mixture of chloroform and methanol (100: 1 v / v, 1.5 liter; 80: 1 v / v, 1.5 liter). A mixture of chloroform and methanol (100: 1 and 80:
The target compound was eluted by 1). Fractions containing the target compound were combined and concentrated under reduced pressure to obtain an oily residue. ODS gel of oily residue (60-200 mesh, Yamamura Chemical Laboratory)
20 ml and the mixture was slurried in ethanol. After evaporation of the solvent, the resulting dry powder is
% ODS gel pre-filled with 3% aqueous methanol (3%
0 ml). After washing with 200 ml of 40% hydrated methanol, hydrated methanol (50
%, 200 ml; 60%, 200 ml). The target compound-containing fractions are combined and concentrated under reduced pressure to give a white powder.
FR901375 substance (206.6 mg) was obtained. This powder was dissolved in hot acetone. The solution was kept at −20 ° C. to obtain a purified FR901375 substance (178 mg) as colorless crystals.

Continuation of the front page (51) Int.Cl. ⁶ identification code FI C12R 1:38) (58) Field surveyed (Int.Cl. ⁶ , DB name) C07K 14/21 C12P 21/00-21/02 A61K 38 / 00-38/58

Claims (1)

(57) [Claims] 1. A FR901375 substance represented by the following formula: